We have demonstrated antigen-specific regression of established murine tumors and resistance to challenge with adoptive immunotherapy (AIT) sing tumor-sensitized T cells activated in vitro with bryostatin 1 and ionomycin (B/I). B/I selectively activates effector/memory cells, which express low levels of L-selection (CD62L). We propose experiments to delineate the cellular and molecular mechanisms that account for these novel observations. The specific aims will address two sets of questions: 1. We will test hypotheses that may explain why B/I selectively activates antigen-sensitized CD62L T cells. We will determine whether differential expression or activation of protein kinase C (PKC) isoforms or other signaling events accounts for this selective activation. Tumor-sensitized T cells separately by CD62L expression and sensitized versus naive T cells from TcR transgenic mice will be used for these studies. 2. Using peptide-specific probes and responses, we will test the hypothesis that adoptive transfer of antigen-sensitized T cells activated in vitro with B/I amplifies immune responses against tumor antigen by increasing the number of antigen-responsive T cells, compared to direct vaccination. We will also test the hypothesis that this amplification can increase the antitumor efficacy of peptide vaccination for treatment of established tumors and spontaneous metastases. We have already shown that AIT with pharmacologically activated T cells from tumor cell-vaccinated mice is more effective than direct vaccination. These studies will be carried out using the 4T07/4T1 murine mammary carcinoma model and the leukemia virus-derived AH1 peptide (SPSYVYHQF) recently described to be a dominant T cell epitope for this tumor. The number of AH1-specific T cells will be enumerated by ELISpot assays and by flow cytometry using commercially available Ld-Ig dimmers and synthetic peptides. We will also determine whether enhancement of AIT antitumor effects by cyclophosphamide pre-treatment reflects increased proliferation of adoptively transferred T cells. We will also test the concept that B/I-activated T cells proliferate in vivo after adoptive transfer by using a vital fluorescent dye to "track" the number of cells and the number of cell division derived from the adoptively transferred cells. Finally, we will test whether AIT with tumor or peptide-sensitized DLN cells activated with B/I will be effective in the adjuvant setting against established spontaneous metastases from 4T1 tumors. These studies will increase our understanding of how to manipulate immune responses to tumor antigens and of basic T lymphocyte biology.